Plant Transformation
In recent years, advances in molecular biology have allowed mankind to manipulate the genetic complement of animals and plants. Genetic engineering of plants entails the isolation and manipulation of genetic material (typically in the form of DNA or RNA) and the subsequent introduction of the genetic material into plants. Such techniques mainly include methods for delivering a nucleic acid into a plant cell or plant tissue to produce transformed cells, and methods of regenerating transgenic plants from the stably transformed cells. Such technology has led to the development of plants with increased pest resistance, herbicide resistance, plants that are capable of expressing pharmaceuticals and other chemicals and plants that express beneficial agricultural traits, such as increased yield, fiber quality and improved digestibility. Advantageously, such plants not only contain genes of interest, but also remain fertile.
One of the most common delivery methods utilizes Agrobacterium-mediated transformation; other frequently used methods involve direct DNA transfer methods (summarized in: Songstad et al., 1995. Plant cell, Tissue and Organ Culture, 40:1-15), including microprojectile bombardment, electroporation of protoplasts or germinating pollen silicon carbide fibers, electrophoresis, polyethylene glycol (PEG) mediated DNA uptake and microinjection. Of these methods, the Agrobacterium-mediated and the microprojectile bombardment methods are routinely used for plant transformation today.
Although the above-described methods have all been shown to be efficient for plant transformation, each has its disadvantages. For Agrobacterium the major disadvantages include its clone dependent infection efficiency, and the need to eliminate the bacteria after the DNA transformation. In the case of microprojectile bombardment, bombardment results in foreign DNA and chromosomes breaking as well as multiple copy insertion, which are undesirable phenomena.
The current microinjection methods utilizing rough needles are problematic methods, because the plant cell wall represents a barrier to the needles while the vacuolar membrane is easily ruptured by such needles, resulting in the leakage of the hydrolytic vacuolar contents and cell death. The technique is also very slow and requires an expensive micromanipulator and substantial manpower resources (Songstad et al., 1995, supra). In addition, the above-described methods are all limited to the delivery of nucleic acids.
Stinging Capsules
Cnidaria (hydras, sea anemones, jellyfish and corals) are aquatic animals, which possess a variety of compounds that are stored and delivered via specialized capsules (cnidocysts). These capsules form a part of specialized cells termed stinging cells (cnidocytes, nematocytes, ptychocytes and the like). The stinging capsules act as microscopic syringes and serve as a predatory or defense mechanism. The Cnidaria family, which encompasses 10,000 known species, includes sedentary single or colonial polyps and pelagic jellyfish. In some of these species, cnidocytes account for more than 45% of the cells present (Tardent 1995. BioEssays, 17(4):351-362). There are a few dozen known types of cnidocysts (also termed cnidae) including more than 30 varieties of nematocysts found in most Cnidaria and spirocysts, and ptychocysts found mainly in the Cnidaria class Anthozoa (Mariscal 1974. In: Coelenterate biology: reviews and new perspectives, Academic Press, New York).
The ability of the stinging cells to penetrate and insert therapeutic or cosmetic agents into mammalian cells including human cells is disclosed in International Patent Application Publication No. WO 02/26191 and corresponding U.S. Pat. No. 6,613,344, fully incorporated herein by reference. A cnidocyst is a hardened dense capsule containing a highly folded inverted tubule and filled with liquid. The tubule may feature a specialized structure such as a shaft, barb, spine, and/or stylet. In nature, the cnidocyst discharges and releases its tubule into tissue following physical or chemical triggering.
Discharge is initiated by a rapid osmotic influx of water, which generates an internal hydrostatic (liquid) pressure of about 150 atmospheres, forcing capsule rupture and ejection of the tubule (Holstein and Tardent 1984. Science, 223(4638): 830-3). During ejection, the long coiled and twisted tubule is averted and its length increases by 95 percent. Accelerating at 40,000 g, the tubule untwists to generate a torque force, which rotates the tubule several times around its axis. These mechanical processes generate a powerful driving force, which enables efficient delivery of the compounds, the toxins and enzymes stored within the capsule (Lotan et al. 1995. Nature, 375(6531):456; Lotan et al. 1996. J Exp Zool, 275(6):444-51; Tardent 1995, supra). This process, which occurs within microseconds, is among the most rapid exocytosis events in biology (Holstein and Tardent 1984, supra).
International Patent Application WO03/079967 discloses methods, compositions and devices utilizing stinging cells containing an exogenous polynucleotide encoding a therapeutic or diagnostic agent or a cosmetic agent for delivering the agent into human cells or tissue. The use of stinging cells has hitherto been demonstrated only for the delivery of therapeutic or cosmetic agents to mammals. Plant cells are different from mammalian cells in that they comprise a rigid cell wall apposed to, and external to the plant protoplast. The plant protoplast comprises a plasma membrane enclosing the cell cytoplasm. The presence of a cell wall gives the plants its rigidity and protects the plant protoplast against outside injuries. However, as described above, the cell wall also limits the number of methods that can be utilized efficiently for the delivery of external biologically active agents into the plant protoplasts.
Thus, there is a recognized need for, and it would be highly advantageous to have efficient, easy to use compositions and methods for the delivery of various biologically active agents into the plant cell or plant tissue.